Live-cell imaging reveals the trade-off between target search flexibility and efficiency for Cas9 and Cas12a.

CRISPR-Cas systems have widely been adopted as genome editing tools, with two frequently employed Cas nucleases being SpyCas9 and LbCas12a. Although both nucleases use RNA guides to find and cleave target DNA sites, the two enzymes differ in terms of protospacer-adjacent motif (PAM) requirements, guide architecture and cleavage mechanism. In the last years, rational engineering led to the creation of PAM-relaxed variants SpRYCas9 and impLbCas12a to broaden the targetable DNA space. By employing their catalytically inactive variants (dCas9/dCas12a), we quantified how the protein-specific characteristics impact the target search process. To allow quantification, we fused these nucleases to the photoactivatable fluorescent protein PAmCherry2.1 and performed single-particle tracking in cells of Escherichia coli. From our tracking analysis, we derived kinetic parameters for each nuclease with a non-targeting RNA guide, strongly suggesting that interrogation of DNA by LbdCas12a variants proceeds faster than that of SpydCas9. In the presence of a targeting RNA guide, both simulations and imaging of cells confirmed that LbdCas12a variants are faster and more efficient in finding a specific target site. Our work demonstrates the trade-off of relaxing PAM requirements in SpydCas9 and LbdCas12a using a powerful framework, which can be applied to other nucleases to quantify their DNA target search.

Temporal analysis of relative distances (TARDIS) is a robust, parameter-free alternative to single-particle tracking.

In single-particle tracking, individual particles are localized and tracked over time to probe their diffusion and molecular interactions. Temporal crossing of trajectories, blinking particles, and false-positive localizations present computational challenges that have remained difficult to overcome. Here we introduce a robust, parameter-free alternative to single-particle tracking: temporal analysis of relative distances (TARDIS). In TARDIS, an all-to-all distance analysis between localizations is performed with increasing temporal shifts. These pairwise distances represent either intraparticle distances originating from the same particle, or interparticle distances originating from unrelated particles, and are fitted analytically to obtain quantitative measures on particle dynamics. We showcase that TARDIS outperforms tracking algorithms, benchmarked on simulated and experimental data of varying complexity. We further show that TARDIS performs accurately in complex conditions characterized by high particle density, strong emitter blinking or false-positive localizations, and is in fact limited by the capabilities of localization algorithms. TARDIS' robustness enables fivefold shorter measurements without loss of information.

Enabling Spectrally Resolved Single-Molecule Localization Microscopy at High Emitter Densities.

Single-molecule localization microscopy (SMLM) is a powerful super-resolution technique for elucidating structure and dynamics in the life- and material sciences. Simultaneously acquiring spectral information (spectrally resolved SMLM, sSMLM) has been hampered by several challenges: an increased complexity of the optical detection pathway, lower accessible emitter densities, and compromised spatio-spectral resolution. Here we present a single-component, low-cost implementation of sSMLM that addresses these challenges. Using a low-dispersion transmission grating positioned close to the image plane, the +1stdiffraction order is minimally elongated and is analyzed using existing single-molecule localization algorithms. The distance between the 0th and 1st order provides accurate information on the spectral properties of individual emitters. This method enables a 5-fold higher emitter density while discriminating between fluorophores whose peak emissions are less than 15 nm apart. Our approach can find widespread use in single-molecule applications that rely on distinguishing spectrally different fluorophores under low photon conditions.

Raw Data to Results: A Hands-On Introduction and Overview of Computational Analysis for Single-Molecule Localization Microscopy.

Single-molecule localization microscopy (SMLM) is an advanced microscopy method that uses the blinking of fluorescent molecules to determine the position of these molecules with a resolution below the diffraction limit (∼5-40 nm). While SMLM imaging itself is becoming more popular, the computational analysis surrounding the technique is still a specialized area and often remains a "black box" for experimental researchers. Here, we provide an introduction to the required computational analysis of SMLM imaging, post-processing and typical data analysis. Importantly, user-friendly, ready-to-use and well-documented code in Python and MATLAB with exemplary data is provided as an interactive experience for the reader, as well as a starting point for further analysis. Our code is supplemented by descriptions of the computational problems and their implementation. We discuss the state of the art in computational methods and software suites used in SMLM imaging and data analysis. Finally, we give an outlook into further computational challenges in the field.

Integrating engineered point spread functions into the phasor-based single-molecule localization microscopy framework.

In single-molecule localization microscopy (SMLM), the use of engineered point spread functions (PSFs) provides access to three-dimensional localization information. The conventional approach of fitting PSFs with a single 2-dimensional Gaussian profile, however, often falls short in analyzing complex PSFs created by placing phase masks, deformable mirrors or spatial light modulators in the optical detection pathway. Here, we describe the integration of PSF modalities known as double-helix, saddle-point or tetra-pod into the phasor-based SMLM (pSMLM) framework enabling fast CPU based localization of single-molecule emitters with sub-pixel accuracy in three dimensions. For the double-helix PSF, pSMLM identifies the two individual lobes and uses their relative rotation for obtaining z-resolved localizations. For the analysis of saddle-point or tetra-pod PSFs, we present a novel phasor-based deconvolution approach entitled circular-tangent pSMLM. Saddle-point PSFs were experimentally realized by placing a deformable mirror in the Fourier plane and modulating the incoming wavefront with specific Zernike modes. Our pSMLM software package delivers similar precision and recall rates to the best-in-class software package (SMAP) at signal-to-noise ratios typical for organic fluorophores and achieves localization rates of up to 15 kHz (double-helix) and 250 kHz (saddle-point/tetra-pod) on a standard CPU. We further integrated pSMLM into an existing software package (SMALL-LABS) suitable for single-particle imaging and tracking in environments with obscuring backgrounds. Taken together, we provide a powerful hardware and software environment for advanced single-molecule studies.

Spatiotemporal Heterogeneity of κ-Carrageenan Gels Investigated via Single-Particle-Tracking Fluorescence Microscopy.

Hydrogels made of the polysaccharide κ-carrageenan are widely used in the food and personal care industry as thickeners or gelling agents. These hydrogels feature dense regions embedded in a coarser bulk network, but the characteristic size and behavior of these regions have remained elusive. Here, we use single-particle-tracking fluorescence microscopy (sptFM) to quantitatively describe κ-carrageenan gels. Infusing fluorescent probes into fully gelated κ-carrageenan hydrogels resulted in two distinct diffusional behaviors. Obstructed self-diffusion of the probes revealed that the coarse network consists of κ-carrageenan strands with a typical diameter of 3.2 ± 0.3 nm leading to a nanoprobe diffusion coefficient of ∼1-5 × 10-12 m2/s. In the dense network regions, we found a fraction with a largely decreased diffusion coefficient of ∼1 × 10-13 m2/s. We also observed dynamic exchange between these states. The computation of spatial mobility maps from the diffusional data indicated that the dense network regions have a characteristic diameter of ∼1 µm and show mobility on the second-to-minute timescale. sptFM provides an unprecedented view of spatiotemporal heterogeneity of hydrogel networks, which we believe bears general relevance for understanding transport and release of both low- and high-molecular weight solutes.

Direct Visualization of Native CRISPR Target Search in Live Bacteria Reveals Cascade DNA Surveillance Mechanism.

CRISPR-Cas systems encode RNA-guided surveillance complexes to find and cleave invading DNA elements. While it is thought that invaders are neutralized minutes after cell entry, the mechanism and kinetics of target search and its impact on CRISPR protection levels have remained unknown. Here, we visualize individual Cascade complexes in a native type I CRISPR-Cas system. We uncover an exponential relation between Cascade copy number and CRISPR interference levels, pointing to a time-driven arms race between invader replication and target search, in which 20 Cascade complexes provide 50% protection. Driven by PAM-interacting subunit Cas8e, Cascade spends half its search time rapidly probing DNA (∼30 ms) in the nucleoid. We further demonstrate that target DNA transcription and CRISPR arrays affect the integrity of Cascade and affect CRISPR interference. Our work establishes the mechanism of cellular DNA surveillance by Cascade that allows the timely detection of invading DNA in a crowded, DNA-packed environment.

Visualisation of dCas9 target search in vivo using an open-microscopy framework.

CRISPR-Cas9 is widely used in genomic editing, but the kinetics of target search and its relation to the cellular concentration of Cas9 have remained elusive. Effective target search requires constant screening of the protospacer adjacent motif (PAM) and a 30 ms upper limit for screening was recently found. To further quantify the rapid switching between DNA-bound and freely-diffusing states of dCas9, we developed an open-microscopy framework, the miCube, and introduce Monte-Carlo diffusion distribution analysis (MC-DDA). Our analysis reveals that dCas9 is screening PAMs 40% of the time in Gram-positive Lactoccous lactis, averaging 17 ± 4 ms per binding event. Using heterogeneous dCas9 expression, we determine the number of cellular target-containing plasmids and derive the copy number dependent Cas9 cleavage. Furthermore, we show that dCas9 is not irreversibly bound to target sites but can still interfere with plasmid replication. Taken together, our quantitative data facilitates further optimization of the CRISPR-Cas toolbox.

Evaluating single-particle tracking by photo-activation localization microscopy (sptPALM) in Lactococcus lactis.

Lactic acid bacteria (LAB) are frequently used in food fermentation and are invaluable for the taste and nutritional value of the fermentation end-product. To gain a better understanding of underlying biochemical and microbiological mechanisms and cell-to-cell variability in LABs, single-molecule techniques such as single-particle tracking photo-activation localization microscopy (sptPALM) hold great promises but are not yet employed due to the lack of detailed protocols and suitable assays. Here, we qualitatively test various fluorescent proteins including variants that are photoactivatable and therefore suitable for sptPALM measurements in Lactococcus lactis, a key LAB for the dairy industry. In particular, we fused PAmCherry2 to dCas9 allowing the successful tracking of single dCas9 proteins, whilst the dCas9 chimeras bound to specific guide RNAs retained their gene silencing ability in vivo. The diffusional information of the dCas9 without any targets showed different mechanistic states of dCas9: freely diffusing, bound to DNA, or transiently interacting with DNA. The capability of performing sptPALM with dCas9 in L. lactis can lead to a better, general understanding of CRISPR-Cas systems as well as paving the way for CRISPR-Cas based interrogations of cellular functions in LABs.

Manipulation of Recrystallization and Network Formation of Oil-Dispersed Micronized Fat Crystals.

A detailed investigation was carried out on the modulation of the coupling between network formation and the recrystallization of oil-dispersed micronized fat crystal (MFC) nanoplatelets by varying oil composition, shear, and temperature. Sunflower (SF) and bean (BO) oils were used as dispersing media for MFC nanoplatelets. During MFC dispersion production at high shear, a significant increase in the average crystal thickness (ACT) could be observed, pointing to recrystallization of the MFC nanoplatelets. More rapid recrystallization of MFC occurred in the SF dispersion than in the BO dispersion, which is attributed to higher solubility of MFC in the SF oil. When the dispersions were maintained under low shear in narrow gap Couette geometry, we witnessed two stages of recrystallization (measured via rheo-SAXD) and the development of a local yield stress (measured via rheo-MRI). In the first stage, shear-enabled mass transfer induces rapid recrystallization of randomly distributed MFC nanoplatelets, which is reflected in a rapid increase in ACT (rheo-SAXD). The formation of a space-filling weak-link MFC network explains the increase in yield stress (assessed in real time by rheo-MRI). In this second stage, recrystallization slows down and yield stress decreases as a result of the formation of MFC aggregates in the weak link network, as observed by confocal Raman imaging. The high fractal dimension of the weak-link network indicates that aggregation takes place via a particle-cluster mechanism. The effects of oil type and shear on the recrystallization rate and network strength could be reproduced in a stirred bowl with a heterogeneous shear stress field, which opens perspectives for the rational manipulation of MFC thickness and network strength under industrial processing conditions.

Phasor based single-molecule localization microscopy in 3D (pSMLM-3D): An algorithm for MHz localization rates using standard CPUs.

We present a fast and model-free 2D and 3D single-molecule localization algorithm that allows more than 3 × 106 localizations per second to be calculated on a standard multi-core central processing unit with localization accuracies in line with the most accurate algorithms currently available. Our algorithm converts the region of interest around a point spread function to two phase vectors (phasors) by calculating the first Fourier coefficients in both the x- and y-direction. The angles of these phasors are used to localize the center of the single fluorescent emitter, and the ratio of the magnitudes of the two phasors is a measure for astigmatism, which can be used to obtain depth information (z-direction). Our approach can be used both as a stand-alone algorithm for maximizing localization speed and as a first estimator for more time consuming iterative algorithms.